EVOLUTION OF DOUBLE MINUTE CHROMOSOMES IN RADIATION INDUCED METHOTREXATE RESISTANT CULTURED MOUSE CELLS

1991 ◽  
pp. 340
Author(s):  
P. HAHN ◽  
B. NEVALDINE ◽  
J. LONGO
Keyword(s):  
Double Minute ◽  
Radiation Induced ◽  
Mouse Cells ◽  
Double Minute Chromosomes

Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells

1984 ◽  
Vol 4 (3) ◽  
pp. 500-506
Author(s):  
S M Robertson ◽  
V Ling ◽  
C P Stanners
Keyword(s):  
Drug Resistance ◽  
Multiple Drug Resistance ◽  
Resistant Mutant ◽  
Genetic System ◽  
Cell Membrane Permeability ◽  
Multiple Drug ◽  
P Glycoprotein ◽  
Double Minute ◽  
Mouse Cells ◽  
Double Minute Chromosomes

A genetic system comprised of mammalian cell mutants which demonstrate concomitant resistance to a number of unrelated drugs has been described previously. The resistance is due to reduced cell membrane permeability and is correlated with the presence of large amounts of a plasma membrane glycoprotein termed P-glycoprotein. This system could represent a model for multiple drug resistance which develops in cancer patients treated with chemotherapeutic drugs. We demonstrate here that the multiple drug resistance phenotype can be transferred to mouse cells with DNA from a drug-resistant mutant and then amplified quantitatively by culture in media containing increasing concentrations of drug. The amount of P-glycoprotein was correlated directly with the degree of drug resistance in the transformants and amplified transformants. In addition, the drug resistance and expression of P-glycoprotein of the transformants were unstable and associated quantitatively with the number of double minute chromosomes. We suggest that the gene for multiple drug resistance and P-glycoprotein is contained in these extrachromosomal particles and is amplified by increases in double minute chromosome number. The potential use of this system for manipulation of mammalian genes in general is discussed.

Molecular structure and evolution of double-minute chromosomes in methotrexate-resistant cultured mouse cells

1992 ◽  
Vol 12 (7) ◽  
pp. 2911-2918
Author(s):  
P J Hahn ◽  
B Nevaldine ◽  
J A Longo
Keyword(s):  
Gel Electrophoresis ◽  
Copy Number ◽  
Pulsed Field Gel Electrophoresis ◽  
Dhfr Gene ◽  
Resistant Mouse ◽  
Pulsed Field ◽  
Double Minute ◽  
Mouse Cells ◽  
Selection For ◽  
Double Minute Chromosomes

To determine whether microscopically visible double-minute chromosomes (DMs) are derived from submicroscopic precursors, we monitored the amplification of the dihydrofolate reductase (DHFR) gene in 10 independent isolates of methotrexate (MTX)-resistant mouse cells. At every other doubling in MTX concentration, the cells were examined both microscopically, to detect the presence of microscopically visible DMs, and by pulsed-field gel electrophoresis and hybridization to a DHFR-specific probe, to detect submicroscopic DMs. One of the cloned MTX-resistant isolates was examined in detail and was shown to originally contain amplified DHFR genes on circular DMs measuring 1 and 3 Mb in size; additionally, metaphase chromosome preparations from this cloned isolate were examined and were shown to contain microscopically visible DMs too large to enter a pulsed-field gel. During stepwise selection for increasing levels of MTX, the smaller DMs (not microscopically visible) were shown to be preferentially amplified, whereas the larger (microscopically visible) ones decreased in relative numbers. Rare-cutting NotI digestion patterns of total genomic DNA that includes the DMs containing the DHFR gene suggest that the DMs increase in copy number without any further significant rearrangements. We saw no evidence from any of the 10 isolates to suggest that microscopically visible DMs are formed from smaller submicroscopic precursors.

scholarly journals Molecular structure and evolution of double-minute chromosomes in methotrexate-resistant cultured mouse cells.

1992 ◽  
Vol 12 (7) ◽  
pp. 2911-2918 ◽  
Author(s):  
P J Hahn ◽  
B Nevaldine ◽  
J A Longo
Keyword(s):  
Gel Electrophoresis ◽  
Copy Number ◽  
Pulsed Field Gel Electrophoresis ◽  
Dhfr Gene ◽  
Resistant Mouse ◽  
Pulsed Field ◽  
Double Minute ◽  
Mouse Cells ◽  
Selection For ◽  
Double Minute Chromosomes

To determine whether microscopically visible double-minute chromosomes (DMs) are derived from submicroscopic precursors, we monitored the amplification of the dihydrofolate reductase (DHFR) gene in 10 independent isolates of methotrexate (MTX)-resistant mouse cells. At every other doubling in MTX concentration, the cells were examined both microscopically, to detect the presence of microscopically visible DMs, and by pulsed-field gel electrophoresis and hybridization to a DHFR-specific probe, to detect submicroscopic DMs. One of the cloned MTX-resistant isolates was examined in detail and was shown to originally contain amplified DHFR genes on circular DMs measuring 1 and 3 Mb in size; additionally, metaphase chromosome preparations from this cloned isolate were examined and were shown to contain microscopically visible DMs too large to enter a pulsed-field gel. During stepwise selection for increasing levels of MTX, the smaller DMs (not microscopically visible) were shown to be preferentially amplified, whereas the larger (microscopically visible) ones decreased in relative numbers. Rare-cutting NotI digestion patterns of total genomic DNA that includes the DMs containing the DHFR gene suggest that the DMs increase in copy number without any further significant rearrangements. We saw no evidence from any of the 10 isolates to suggest that microscopically visible DMs are formed from smaller submicroscopic precursors.

scholarly journals Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells.

1984 ◽  
Vol 4 (3) ◽  
pp. 500-506 ◽  
Author(s):  
S M Robertson ◽  
V Ling ◽  
C P Stanners
Keyword(s):  
Drug Resistance ◽  
Multiple Drug Resistance ◽  
Resistant Mutant ◽  
Genetic System ◽  
Cell Membrane Permeability ◽  
Multiple Drug ◽  
P Glycoprotein ◽  
Double Minute ◽  
Mouse Cells ◽  
Double Minute Chromosomes

A genetic system comprised of mammalian cell mutants which demonstrate concomitant resistance to a number of unrelated drugs has been described previously. The resistance is due to reduced cell membrane permeability and is correlated with the presence of large amounts of a plasma membrane glycoprotein termed P-glycoprotein. This system could represent a model for multiple drug resistance which develops in cancer patients treated with chemotherapeutic drugs. We demonstrate here that the multiple drug resistance phenotype can be transferred to mouse cells with DNA from a drug-resistant mutant and then amplified quantitatively by culture in media containing increasing concentrations of drug. The amount of P-glycoprotein was correlated directly with the degree of drug resistance in the transformants and amplified transformants. In addition, the drug resistance and expression of P-glycoprotein of the transformants were unstable and associated quantitatively with the number of double minute chromosomes. We suggest that the gene for multiple drug resistance and P-glycoprotein is contained in these extrachromosomal particles and is amplified by increases in double minute chromosome number. The potential use of this system for manipulation of mammalian genes in general is discussed.

Induction of metallothionein-I mRNA in cultured cells by heavy metals and iodoacetate: evidence for gratuitous inducers

1984 ◽  
Vol 4 (3) ◽  
pp. 484-491
Author(s):  
D M Durnam ◽  
R D Palmiter
Keyword(s):  
Heavy Metals ◽  
Cultured Cells ◽  
Mrna Accumulation ◽  
Mouse Hepatocyte ◽  
Hepatocyte Cell Line ◽  
Regulatory Molecule ◽  
Heavy Metal Detoxification ◽  
Double Minute ◽  
Double Minute Chromosomes

A mouse hepatocyte cell line selected for growth in 80 microM CdSO4 (Cdr80 cells) was used to test the role of metallothioneins in heavy metal detoxification. The cadmium-resistant (Cdr80) cells have double minute chromosomes carrying amplified copies of the metallothionein-I gene and accumulate ca. 20-fold more metallothionein-I mRNA than unselected cadmium-sensitive (Cds) cells after optimal Cd stimulation. As a consequence, the amount of Cd which inhibits DNA synthesis by 50% is ca. 7.5-fold higher in Cdr80 cells than in Cds cells. Cds and Cdr80 cells were compared in terms of their resistance to other heavy metals. The results indicate that although Zn, Cu, Hg, Ag, Co, Ni, and Bi induce metallothionein-I mRNA accumulation in both Cdr80 and Cds cells, the Cdr80 cells show increased resistance to only a subset of these metals (Zn, Cu, Hg, and Bi). This suggests that not all metals which induce metallothionein mRNA are detoxified by metallothionein and argues against autoregulation of metallothionein genes. Metallothionein-I mRNA is also induced by iodoacetate, suggesting that the regulatory molecule has sensitive sulfhydryl groups.

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